Multiplex Dual-Target Reverse Transcription PCR for Subtyping Avian Influenza A(H5) Virus

An increased risk for human infection with avian influenza A(H5N1) viruses is of concern. We developed an internally controlled, dual-target reverse transcription PCR for influenza A(H5) subtyping. This test could be used to detect influenza A(H5) in clinical samples.


DISPATCHES
H ighly pathogenic avian influenza (HPAI) virus- es cause high mortality rates in wild birds and farmed poultry, and their potential for adaptation to humans remains a major pandemic threat (1).In 2021, a notable increase in avian influenza activity occurred, driven by the emergence of influenza A(H5N1) clade 2.3.4.4b viruses (2,3).Suspected mammal-to-mammal transmission during outbreaks among seals and minks led to concerns regarding the escalation of risk to humans (4)(5)(6)(7).In 2024, the virus was identified in dairy cows in the United States (8), and exposure to infected cows has resulted in transmission to humans (9).To prepare for possible additional human cases, we developed an internally controlled, dual-target H5 subtyping quantitative reverse transcription PCR (qRT-PCR) based on primer-probe sequences from the World Health Organization (10).We evaluated this qRT-PCR by using synthesized nucleic acids, cultured avian and human virus isolates, and clinical upper respiratory specimens collected from patients with influenza A virus infection.

The Study
We identified previously published primer-probe sets from the World Health Organization that target different regions of the influenza A(H5) hemagglutinin (HA) gene for combination into a dual-target H5 subtyping qRT-PCR (Appendix Table 1, https:// wwwnc.cdc.gov/EID/article/30/8/24-0785-App1.pdf) (10).We introduced minor sequence changes to account for recent clade 2.3.4.4b diversity, and simultaneously, to limit primer-probe complexity (Table 1).We evaluated these primers and probes against complete North American H5 clade 2.3.4.4bHA gene sequences in the GISAID database (https://www.gisaid.org)during January 1, 2022-May 29, 2024, and found that 99.8% (5,975/5,987) of sequences aligned with a maximum of 1 mismatch to primer-probe set 1 and 97.7% (5,972/5,990) aligned with a maximum of 1 mismatch to primer-probe set 2 (Appendix Tables 2, 3).We observed no primer-probe mismatches compared with the influenza A H5 sequences obtained from persons with H5 infections in the United States (4 persons as of May 29, 2024) (Appendix Tables 4,  5).We combined the primer-probe sets in multiplex with primers-probes for the influenza A matrix (M) gene for pan-influenza A detection and with primers-probes for RNase P as an internal control (11).
We determined the 95% lower limit of detection (LLOD) by using single-stranded DNA (ssDNA) encoding the H5 target sequences from HPAI virus clade 1 (GenBank accession no.JQ966928, A/condor/Guangdong/139/2003 [H5N1]) (12) and clade 2.3.4.4b (GenBank accession no.OP499866, A/redtailed hawk/Kansas/W22-198/2022 [H5N1]) viruses (Appendix Table 6).The primer-probe target regions of this clade 2.3.4.4bH5 sequence are identical to the recent H5 sequences from dairy cows and humans.We combined the 2 clade 1 ssDNA targets at equal copy numbers; we did the same with the clade 2.3.4.4b ssDNA targets.We also combined these H5 clade mixes at equal copy numbers with ssDNA encoding the pan-influenza A M gene target.We then diluted the clade 1 ssDNA mix and clade 2.An increased risk for human infection with avian influenza A(H5N1) viruses is of concern.We developed an internally controlled, dual-target reverse transcription PCR for influenza A(H5) subtyping.This test could be used to detect influenza A(H5) in clinical samples.
We then evaluated the H5 subtyping qRT-PCR by using genomic RNA from additional H5 viruses, including 5 low pathogenicity avian influenza non-Gs/Gd isolates from the United States (Table 2).We tested each eluate in duplicate.Although we detected H5 RNA in all reactions, the difference in mean cycle threshold (Ct) values between the H5 and pan-influenza A (M gene) targets varied widely (range 0.6-9.9cycles).Testing the H5 primer-probe sets individually revealed that the US non-Gs/Gd genomic RNAs were detected only by primer-probe set 1. The A/ mallard/Minnesota/16-041335-3/2016 (H5N2) eluate showed the greatest Ct difference, consistent with the highest number of H5 primer-probe mismatches in primer-probe set 1 (9 mismatches) (Appendix Tables 5, 6).
To determine specificity, we tested genomic RNA from cultured human and avian virus isolates encoding non-H5 HA genes (Table 3).We detected the paninfluenza A target in all eluates; median Ct value was 17.3 (range 13.6-20.7).We did not detect the H5 target in any eluates.
We performed further specificity experiments by using residual upper respiratory swab samples in viral transport media submitted for clinical respiratory virus testing.We conducted this work with Stanford Institutional Review Board approval (protocol no.68234); individual consent was waived.We evaluated 100 samples collected

Conclusions
HPAI H5 viruses, particularly clade 2.3.4.4b viruses, pose an emerging pandemic threat.This internally controlled dual-target influenza A(H5) qRT-PCR demonstrated high analytical performance and could be used to directly test samples from patients with suspected HPAI A(H5) virus infection or as a secondary test to subtype known influenza A-positive samples after routine respiratory virus testing.The dual-target design reduces the likelihood of false negatives, as evidenced by detection of low pathogenicity avian influenza H5 viruses with substantial target mismatch.However, continuous sequence surveillance and updating of the primer-probe sets will be required to ensure that this assay accounts for ongoing viral evolution (13).

Table 2 .
Avian influenza A(H5) virus genomic RNA used to evaluate inclusivity in study of multiplex dual-target reverse transcription PCR for subtyping avian influenza A(H5) virus* from the US Department of Agriculture.The genomic RNA from these influenza A(H5) viruses was diluted 1:10 buffer AVE plus carrier RNA (QIAGEN, https://www.qiagen.com)and tested in duplicate.Ct values are means.Am-non-Gs/Gd, non-goose/GuangDong from the United States; Ct, cycle threshold; EA-non-Gs/Gd, Eurasian non-goose/GuangDong; M, matrix gene; NA, not available.
*Kilbourne F181 genomic RNA was obtained from BEI Resources (https://www.beiresources.org).Am-non-Gs/GD A(H5) genomic RNA was obtained t 34.8) by the H5 multiplex.None of those influenza A-positive samples had detectable H5 RNA.Of the respiratory virus panel negative samples, all had detectable RNase P; median Ct value was 23.9 (range 17.7-29.8).None of the panel negative samples had detectable H5 RNA.